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In complex tissues containing cells that are difficult to dissociate, single-nucleus RNA-sequencing (snRNA-seq) has become the preferred experimental technology over single-cell RNA-sequencing (scRNA-seq) to measure gene expression. To accurately model these data in downstream analyses, previous work has shown that droplet-based scRNA-seq data are not zero-inflated, but whether droplet-based snRNA-seq data follow the same probability distributions has not been systematically evaluated. Using pseudonegative control data from nuclei in mouse cortex sequenced with the 10x Genomics Chromium system and mouse kidney sequenced with the DropSeq system, we found that droplet-based snRNA-seq data follow a negative binomial distribution, suggesting that parametric statistical models applied to scRNA-seq are transferable to snRNA-seq. Furthermore, we found that the quantification choices in adapting quantification mapping strategies from scRNA-seq to snRNA-seq can play a significant role in downstream analyses and biological interpretation. In particular, reference transcriptomes that do not include intronic regions result in significantly smaller library sizes and incongruous cell type classifications. We also confirmed the presence of a gene length bias in snRNA-seq data, which we show is present in both exonic and intronic reads, and investigate potential causes for the bias., (© The Author(s) 2023. Published by Oxford University Press.)

Hi-C data are commonly normalized using single sample processing methods, with focus on comparisons between regions within a given contact map. Here, we aim to compare contact maps across different samples. We demonstrate that unwanted variation, of likely technical origin, is present in Hi-C data with replicates from different individuals, and that properties of this unwanted variation change across the contact map. We present band-wise normalization and batch correction, a method for normalization and batch correction of Hi-C data and show that it substantially improves comparisons across samples, including in a quantitative trait loci analysis as well as differential enrichment across cell types., (© The Author(s) 2024. Published by Oxford University Press.)

Summary: Thousands of DNA methylation (DNAm) array samples from human blood are publicly available on the Gene Expression Omnibus (GEO), but they remain underutilized for experiment planning, replication and cross-study and cross-platform analyses. To facilitate these tasks, we augmented our recountmethylation R/Bioconductor package with 12 537 uniformly processed EPIC and HM450K blood samples on GEO as well as several new features. We subsequently used our updated package in several illustrative analyses, finding (i) study ID bias adjustment increased variation explained by biological and demographic variables, (ii) most variation in autosomal DNAm was explained by genetic ancestry and CD4+ T-cell fractions and (iii) the dependence of power to detect differential methylation on sample size was similar for each of peripheral blood mononuclear cells (PBMC), whole blood and umbilical cord blood. Finally, we used PBMC and whole blood to perform independent validations, and we recovered 38-46% of differentially methylated probes between sexes from two previously published epigenome-wide association studies., Availability and Implementation: Source code to reproduce the main results are available on GitHub (repo: recountmethylation_flexible-blood-analysis_manuscript; url: https://github.com/metamaden/recountmethylation_flexible-blood-analysis_manuscript). All data was publicly available and downloaded from the Gene Expression Omnibus (https://www.ncbi.nlm.nih.gov/geo/). Compilations of the analyzed public data can be accessed from the website recount.bio/data (preprocessed HM450K array data: https://recount.bio/data/remethdb_h5se-gm_epic_0-0-2_1589820348/; preprocessed EPIC array data: https://recount.bio/data/remethdb_h5se-gm_epic_0-0-2_1589820348/)., Supplementary Information: Supplementary data are available at Bioinformatics Advances online., (© The Author(s) 2023. Published by Oxford University Press.)

While DNA methylation (DNAm) is the most-studied epigenetic mark, few recent studies probe the breadth of publicly available DNAm array samples. We collectively analyzed 35 360 Illumina Infinium HumanMethylation450K DNAm array samples published on the Gene Expression Omnibus. We learned a controlled vocabulary of sample labels by applying regular expressions to metadata and used existing models to predict various sample properties including epigenetic age. We found approximately two-thirds of samples were from blood, one-quarter were from brain and one-third were from cancer patients. About 19% of samples failed at least one of Illumina's 17 prescribed quality assessments; signal distributions across samples suggest modifying manufacturer-recommended thresholds for failure would make these assessments more informative. We further analyzed DNAm variances in seven tissues (adipose, nasal, blood, brain, buccal, sperm and liver) and characterized specific probes distinguishing them. Finally, we compiled DNAm array data and metadata, including our learned and predicted sample labels, into database files accessible via the recountmethylation R/Bioconductor companion package. Its vignettes walk the user through some analyses contained in this paper., (© The Author(s) 2021. Published by Oxford University Press on behalf of NAR Genomics and Bioinformatics.)

Objectives: To compare condylar development and changes in condylar radiological appearance in patients with idiopathic condylar resorption (ICR) to a healthy, age- and gender matched, control group., Materials and Methods: This case-control study included 16 ICR patients [mean age: 15years, 9 months; standard deviation (SD): 4 years) and 16 controls (mean age: 16 years, 8 months; SD: 4 years, 7 months), with diagnostic (T0) and 2-year follow-up (T1) CBCT examinations. Condylar changes were evaluated through changes in condylar neck angle (CNA), and the transversal, vertical and anteroposterior displacement of five condylar points between T0 and T1. The magnitude and direction of condylar changes were evaluated using vector analyses. A qualitative radiological evaluation of the TMJ was performed based on healthy, erosive and repaired morphological appearance. Linear and angular measurements were assessed using ANOVA and a Tukey post-hoc test, and vectors were tested using an independent-sample 2-tailed t-test. Fisher's exact test was used for the qualitative evaluation., Results: At T0, ICR patients exhibited decreased condylar height, smaller condylar width and posteriorly inclined CNA compared with the control group (P < 0.05). During observation, condylar vertical growth was smaller in the ICR group than in the control group (P < 0.05). Vector analysis showed an upward direction of vertical displacement for all condylar points in the control group; the ICR group showed a downward direction (P < 0.003). The radiological appearance of 75% of the ICR condyles and 94% of the control condyles did not change during the 2-year follow-up period., Conclusions: ICR condyles displayed reduced vertical development compared with control condyles. The radiological appearance remained unchanged for most joints. Observed changes in radiological appearance did not always follow a progressive model of degenerative joint disease., (© The Author(s) 2020. Published by Oxford University Press on behalf of the European Orthodontic Society. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

In the study of DNA methylation, genetic variation between species, strains or individuals can result in CpG sites that are exclusive to a subset of samples, and insertions and deletions can rearrange the spatial distribution of CpGs. How to account for this variation in an analysis of the interplay between sequence variation and DNA methylation is not well understood, especially when the number of CpG differences between samples is large. Here, we use whole-genome bisulfite sequencing data on two highly divergent mouse strains to study this problem. We show that alignment to personal genomes is necessary for valid methylation quantification. We introduce a method for including strain-specific CpGs in differential analysis, and show that this increases power. We apply our method to a human normal-cancer dataset, and show this improves accuracy and power, illustrating the broad applicability of our approach. Our method uses smoothing to impute methylation levels at strain-specific sites, thereby allowing strain-specific CpGs to contribute to the analysis, while accounting for differences in the spatial occurrences of CpGs. Our results have implications for joint analysis of genetic variation and DNA methylation using bisulfite-converted DNA, and unlocks the use of personal genomes for addressing this question., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The incidence of diet-induced metabolic disease has soared over the last half-century, despite national efforts to improve health through universal dietary recommendations. Studies comparing dietary patterns of populations with health outcomes have historically provided the basis for healthy diet recommendations. However, evidence that population-level diet responses are reliable indicators of responses across individuals is lacking. This study investigated how genetic differences influence health responses to several popular diets in mice, which are similar to humans in genetic composition and the propensity to develop metabolic disease, but enable precise genetic and environmental control. We designed four human-comparable mouse diets that are representative of those eaten by historical human populations. Across four genetically distinct inbred mouse strains, we compared the American diet's impact on metabolic health to three alternative diets (Mediterranean, Japanese, and Maasai/ketogenic). Furthermore, we investigated metabolomic and epigenetic alterations associated with diet response. Health effects of the diets were highly dependent on genetic background, demonstrating that individualized diet strategies improve health outcomes in mice. If similar genetic-dependent diet responses exist in humans, then a personalized, or "precision dietetics," approach to dietary recommendations may yield better health outcomes than the traditional one-size-fits-all approach., (Copyright © 2018 by the Genetics Society of America.)

Summary: The minfi package is widely used for analyzing Illumina DNA methylation array data. Here we describe modifications to the minfi package required to support the HumanMethylationEPIC ('EPIC') array from Illumina. We discuss methods for the joint analysis and normalization of data from the HumanMethylation450 ('450k') and EPIC platforms. We introduce the single-sample Noob ( ssNoob ) method, a normalization procedure suitable for incremental preprocessing of individual methylation arrays and conclude that this method should be used when integrating data from multiple generations of Infinium methylation arrays. We show how to use reference 450k datasets to estimate cell type composition of samples on EPIC arrays. The cumulative effect of these updates is to ensure that minfi provides the tools to best integrate existing and forthcoming Illumina methylation array data., Availability and Implementation: The minfi package version 1.19.12 or higher is available for all platforms from the Bioconductor project., Contact: khansen@jhsph.edu., Supplementary Information: Supplementary data are available at Bioinformatics online., (© The Author 2016. Published by Oxford University Press.)

Motivation: Public archives contain thousands of trillions of bases of valuable sequencing data. More than 40% of the Sequence Read Archive is human data protected by provisions such as dbGaP. To analyse dbGaP-protected data, researchers must typically work with IT administrators and signing officials to ensure all levels of security are implemented at their institution. This is a major obstacle, impeding reproducibility and reducing the utility of archived data., Results: We present a protocol and software tool for analyzing protected data in a commercial cloud. The protocol, Rail-dbGaP, is applicable to any tool running on Amazon Web Services Elastic MapReduce. The tool, Rail-RNA v0.2, is a spliced aligner for RNA-seq data, which we demonstrate by running on 9662 samples from the dbGaP-protected GTEx consortium dataset. The Rail-dbGaP protocol makes explicit for the first time the steps an investigator must take to develop Elastic MapReduce pipelines that analyse dbGaP-protected data in a manner compliant with NIH guidelines. Rail-RNA automates implementation of the protocol, making it easy for typical biomedical investigators to study protected RNA-seq data, regardless of their local IT resources or expertise., Availability and Implementation: Rail-RNA is available from http://rail.bio Technical details on the Rail-dbGaP protocol as well as an implementation walkthrough are available at https://github.com/nellore/rail-dbgap Detailed instructions on running Rail-RNA on dbGaP-protected data using Amazon Web Services are available at http://docs.rail.bio/dbgap/, Contacts: : anellore@gmail.com or langmea@cs.jhu.edu, Supplementary Information: Supplementary data are available at Bioinformatics online., (© The Author 2016. Published by Oxford University Press.)

Molecular interrogation of a biological sample through DNA sequencing, RNA and microRNA profiling, proteomics and other assays, has the potential to provide a systems level approach to predicting treatment response and disease progression, and to developing precision therapies. Large publicly funded projects have generated extensive and freely available multi-assay data resources; however, bioinformatic and statistical methods for the analysis of such experiments are still nascent. We review multi-assay genomic data resources in the areas of clinical oncology, pharmacogenomics and other perturbation experiments, population genomics and regulatory genomics and other areas, and tools for data acquisition. Finally, we review bioinformatic tools that are explicitly geared toward integrative genomic data visualization and analysis. This review provides starting points for accessing publicly available data and tools to support development of needed integrative methods., (© The Author 2015. Published by Oxford University Press.)

RNA-sequencing (RNA-seq) is a flexible technology for measuring genome-wide expression that is rapidly replacing microarrays as costs become comparable. Current differential expression analysis methods for RNA-seq data fall into two broad classes: (1) methods that quantify expression within the boundaries of genes previously published in databases and (2) methods that attempt to reconstruct full length RNA transcripts. The first class cannot discover differential expression outside of previously known genes. While the second approach does possess discovery capabilities, statistical analysis of differential expression is complicated by the ambiguity and variability incurred while assembling transcripts and estimating their abundances. Here, we propose a novel method that first identifies differentially expressed regions (DERs) of interest by assessing differential expression at each base of the genome. The method then segments the genome into regions comprised of bases showing similar differential expression signal, and then assigns a measure of statistical significance to each region. Optionally, DERs can be annotated using a reference database of genomic features. We compare our approach with leading competitors from both current classes of differential expression methods and highlight the strengths and weaknesses of each. A software implementation of our method is available on github (https://github.com/alyssafrazee/derfinder)., (© The Author 2014. Published by Oxford University Press.)

The increasing prevalence of antibiotic-resistant bacteria portends an impending postantibiotic age, characterized by diminishing efficacy of common antibiotics and routine application of multifaceted, complementary therapeutic approaches to treat bacterial infections, particularly multidrug-resistant organisms. The first line of defense for most bacterial pathogens consists of a physical and immunologic barrier known as the capsule, commonly composed of a viscous layer of carbohydrates that are covalently bound to the cell wall in Gram-positive bacteria or often to lipids of the outer membrane in many Gram-negative bacteria. Bacterial capsular polysaccharides are a diverse class of high molecular weight polysaccharides contributing to virulence of many human pathogens in the gut, respiratory tree, urinary tract, and other host tissues, by hiding cell surface components that might otherwise elicit host immune response. This review highlights capsular polysaccharides that are structurally identical or similar to polysaccharides found in mammalian tissues, including polysialic acid and glycosaminoglycan capsules hyaluronan, heparosan, and chondroitin. Such nonimmunogenic coatings render pathogens insensitive to certain immune responses, effectively increasing residence time in host tissues and enabling pathologically relevant population densities to be reached. Biosynthetic pathways and capsular involvement in immune system evasion are described, providing a basis for potential therapies aimed at supplementing or replacing antibiotic treatment., (© 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.)

Motivation: The recently released Infinium HumanMethylation450 array (the '450k' array) provides a high-throughput assay to quantify DNA methylation (DNAm) at ∼450 000 loci across a range of genomic features. Although less comprehensive than high-throughput sequencing-based techniques, this product is more cost-effective and promises to be the most widely used DNAm high-throughput measurement technology over the next several years., Results: Here we describe a suite of computational tools that incorporate state-of-the-art statistical techniques for the analysis of DNAm data. The software is structured to easily adapt to future versions of the technology. We include methods for preprocessing, quality assessment and detection of differentially methylated regions from the kilobase to the megabase scale. We show how our software provides a powerful and flexible development platform for future methods. We also illustrate how our methods empower the technology to make discoveries previously thought to be possible only with sequencing-based methods., Availability and Implementation: http://bioconductor.org/packages/release/bioc/html/minfi.html., Contact: khansen@jhsph.edu; rafa@jimmy.harvard.edu, Supplementary Information: Supplementary data are available at Bioinformatics online.

Unilateral temporomandibular joint (TMJ) arthritis in juvenile idiopathic arthritis (JIA) patients often induces asymmetric dentofacial growth. This study evaluates the effects of an orthopaedic functional appliance worn full time to reduce asymmetric mandibular growth in JIA patients. Twenty-two JIA patients with unilateral TMJ involvement were included in the study (mean age 7.5 years, range: 3.8-13.8 years). They all received orthopaedic treatment with a functional appliance, a so-called distraction splint (mean treatment time 57 months). Panoramic tomograms or cone beam-computerized tomograms were taken before and within 12 months after treatment cessation. At both time points, the ratio between the healthy and the affected side of the mandible was evaluated in terms of inter-side differences in condylar height, ramus height, and total vertical mandibular height. Orthopaedic functional treatment reduced mandibular asymmetries in terms of ramus height and total vertical mandibular height (P < 0.05). Mandibular growth rates in the affected and the non-affected sides were comparable in most patients. Our study finds evidence to support that a distraction splint can normalize mandibular vertical growth in the affected side. We therefore suggest implementation of distraction splint therapy in the treatment of JIA patients with unilateral TMJ arthritis.

The ability to measure gene expression on a genome-wide scale is one of the most promising accomplishments in molecular biology. Microarrays, the technology that first permitted this, were riddled with problems due to unwanted sources of variability. Many of these problems are now mitigated, after a decade's worth of statistical methodology development. The recently developed RNA sequencing (RNA-seq) technology has generated much excitement in part due to claims of reduced variability in comparison to microarrays. However, we show that RNA-seq data demonstrate unwanted and obscuring variability similar to what was first observed in microarrays. In particular, we find guanine-cytosine content (GC-content) has a strong sample-specific effect on gene expression measurements that, if left uncorrected, leads to false positives in downstream results. We also report on commonly observed data distortions that demonstrate the need for data normalization. Here, we describe a statistical methodology that improves precision by 42% without loss of accuracy. Our resulting conditional quantile normalization algorithm combines robust generalized regression to remove systematic bias introduced by deterministic features such as GC-content and quantile normalization to correct for global distortions.

Generation of cDNA using random hexamer priming induces biases in the nucleotide composition at the beginning of transcriptome sequencing reads from the Illumina Genome Analyzer. The bias is independent of organism and laboratory and impacts the uniformity of the reads along the transcriptome. We provide a read count reweighting scheme, based on the nucleotide frequencies of the reads, that mitigates the impact of the bias.

The aim of this investigation was to study the effect of intra-articular (i.a.) corticosteroid injections (IACIs) in the temporomandibular joint (TMJ) on mandibular development in antigen-induced TMJ arthritis. Ten-week-old female New Zealand white rabbits (n = 42) were randomly divided into four groups: group A, control (no injections); group B, placebo (repeated i.a. TMJ saline injections); group C, untreated arthritis (repeated induction of TMJ arthritis); and group D, steroid (repeated induction of TMJ arthritis + IACI). All animals had two tantalum implants inserted in the right side of the mandible serving as stable landmarks for later growth analysis. One implant was inserted close to the symphysis and one in the molar region. Computerized tomographic (CT) full-head scans were carried out at 14 (T1) and 26 (T2) weeks of age. (Dropout of animals at T2; group C, n = 7, and group D, n = 3.) Absolute and relative intra- and inter-group growth variations were evaluated during the growth period by comparison of CT scans. One-way analysis of variance was used for T1 statistical analysis, and absolute intra-group and relative inter-group growth differences between T1 and T2 were evaluated by Student's t-tests. At T2, the animals in the group A had greater sagittal and vertical mandibular growth compared with the other three groups. TMJ arthritis caused diminished mandibular growth. However, relative mandibular growth was significantly less in group D. The findings of this study do not indicate a positive long-term effect in the use of IACI in the TMJ as an early treatment intervention against TMJ inflammation in growing individuals.

Aims: The aim of the study was the development of a sensitive human-specific quantitative real-time PCR assay for microbial faecal source tracking (MST) in alpine spring water. The assay detects human-specific faecal DNA markers (BacH) from 16S rRNA gene sequences from the phylum Bacteroidetes using TaqMan minor groove binder probes., Methods and Results: The qualitative and quantitative detection limits of the PCR assay were 6 and 30 marker copies, respectively. Specificity was proved by testing 41 human faeces and waste water samples and excluding cross-amplification from 302 animal faecal samples from Eastern Austria. Marker concentrations in human faecal material were in the range from 6.6 x 10(9) to 9.1 x 10(10) marker equivalents per gram. The method was sensitive enough to detect a few 100 pg of faeces in faecal suspensions. The assay was applied on water samples from an alpine karstic spring catchment area and the results reflected the expected levels of human faecal influence., Conclusions: The method exhibited sufficient sensitivity to allow quantitative source tracking of human faecal impact in the investigated karstic spring water., Significance and Impact of the Study: The developed method constitutes the first quantitative human-specific MST tool sensitive enough for investigations in ground and spring water.

An estimated 15% of invasive group B streptococcal (GBS) disease is caused by type II capsular polysaccharide (II CPS). In developing a pentavalent vaccine for the prevention of GBS infections, individual GBS CPSs have been coupled to tetanus toxoid (TT) to prepare vaccines with enhanced immunogenicity. Type II GBS (GBS II) vaccine was created by direct, covalent coupling of II CPS to TT by reductive amination. In 2 clinical trials, 75 healthy nonpregnant women 18-45 years old were randomized to receive II CPS-TT (II-TT) conjugate (dose range, 3.6-57 microg of CPS component) or uncoupled II CPS vaccine. Both vaccines were well tolerated. II CPS-specific IgG serum concentrations (as well as IgM and IgA) peaked 2 weeks after immunization, being significantly higher in recipients of conjugated vaccine than in recipients of uncoupled CPS. Immunological responses to conjugate were dose dependent and correlated with opsonophagocytosis in vitro. These results support inclusion of II-TT conjugate when preparing a multivalent GBS vaccine.

Two animal models were used to study maternal transfer of antibody to a group B Streptococcus (GBS) type III polysaccharide-tetanus toxoid (III-TT) conjugate. The III-TT vaccine protected all 27 mouse pups born to vaccinated dams against a GBS challenge. In a separate study of vaccinated mouse dams and pups, maternal sera contained all 4 subclasses of polysaccharide-specific IgG, with IgG1 accounting for 83% of total IgG. Specific IgG subclass distribution (IgG1>>IgG2a=IgG2b=IgG3) in newborn pups closely resembled that in their mothers. Seven of 9 female baboons given the III-TT vaccine had 5- to 36-fold increases in specific antibody from baseline levels; they transferred 26%-185% of specific antibody to their offspring. Matched maternal and neonatal sera obtained at delivery were functionally equivalent in an in vitro opsonophagocytosis assay. These preclinical studies provide further evidence for effective immunogenicity of GBS conjugate vaccine and efficient transport of functionally active maternal antibody.

Group B Streptococcus (GBS) types VI and VIII are prevalent among serotypes isolated from pregnant women in Japan. Maternal vaccination with a safe and effective GBS vaccine has been proposed as a rational approach to prevent neonatal GBS disease. Because antibody specific for the capsular polysaccharide (CPS) antigens of GBS is protective, vaccines were developed with purified type VI and VIII CPS coupled to tetanus toxoid. In rabbits the newly synthesized conjugate vaccines elicited high-titered, type-specific antibody that was opsonically active in vitro. Moreover, litters born to mice actively vaccinated with the conjugate vaccines, in contrast to uncoupled CPS or saline, were protected against an ordinarily lethal challenge of GBS of homologous serotype. GBS types VI and VIII conjugate vaccines of the design presented may be important components of a multivalent GBS vaccine for use in regions where these serotypes predominate.

Infection by group B streptococcus (GBS) is an important cause of bacterial disease in neonates, pregnant women, and nonpregnant adults. Whereas serotypes Ia, Ib, II, III, and V are most commonly associated with colonization and disease in the United States, strains of other serotypes have been isolated from patients in Japan. By use of an inhibition ELISA, the serotypes of 73 vaginal colonizing GBS strains isolated from healthy pregnant Japanese women were investigated. Twenty-six (35.6%) were type VIII, 18 (24.7%) were type VI, and the remaining 29 were distributed among more traditional serotypes. Strains were also tested by immunoblot for the presence of GBS surface proteins. Fifty-three (72.6%) of the 73 strains expressed one or more laddering GBS proteins. These data show that type VI and VIII GBS strains are common vaginal isolates in pregnant Japanese women and that one or more laddering proteins are present in most GBS strains.

About 40% of invasive group B streptococcal (GBS) isolates are capsular polysaccharide (CPS) types Ia or Ib. Because infant and maternal GBS infections may be preventable by maternal vaccination, individual GBS CPS have been coupled to tetanus toxoid (TT) to prepare vaccines with enhanced immunogenicity. Immunogenicity in rabbits and protective capacity in mice of a series of type Ia- and Ib-TT conjugates increased with the degree of polysaccharide-to-protein cross-linking. In total, 190 healthy, nonpregnant women aged 18-40 years were randomized in four trials to receive Ia- or Ib-TT conjugate (dose range, 3.75-63 microg of CPS component), uncoupled Ia or Ib CPS, or saline. All vaccines were well-tolerated. CPS-specific IgG serum concentrations peaked 4-8 weeks after vaccination and were significantly higher in recipients of conjugated than of uncoupled CPS. Immune responses to the conjugates were dose-dependent and correlated in vitro with opsonophagocytosis. These results support inclusion of Ia- and Ib-TT conjugates when formulating a multivalent GBS vaccine.

Nonpregnant adults with group B streptococcus bacteremia were identified by active surveillance in three hospitals. Serum samples collected within 2 days of the time of blood culture were assayed for IgG antibodies to the capsular polysaccharide of the infecting strain: serotype Ia (3 isolates), III (5 isolates), or V (4 isolates). In 7 of 12 bacteremia episodes, the serum level of IgG to the infecting isolate was > or = 3.5 microg/mL, higher than the 1-2 microg/mL level thought to be protective in neonates. Among selected acute-phase sera, 4 of 5 that contained > or = 3.5 microg/mL specific IgG mediated efficient opsonophagocytic killing of the corresponding group B streptococcus isolate in vitro. High levels of specific antibodies during the acute phase of invasive group B streptococcus infection in nonpregnant adults may reflect a rapid antibody response to infection or, in some cases, may indicate that susceptibility is due to defects in other immune effectors.

Two immunomodulating polysaccharides, poly-(1-6)-beta-glucotriosyl-(1-3)-beta-glucopyranose (PGG)-glucan and Bacteroides fragilis polysaccharide A (PS A), were evaluated for the prevention of mortality and abscess formation associated with experimental intraabdominal sepsis. Prophylactic treatment with a combination of these compounds significantly reduced mortality (8% vs. 44% in the saline-treated control group) and the incidence of abscesses (30% vs. 100% in the saline-treated control group) after challenge with rat cecal contents. These compounds were also effective when administered therapeutically after bacterial contamination of the peritoneal cavity. PS A treatment conferred long-term protection against abscess formation and resulted in significantly fewer total aerobes and anaerobes in the peritoneal fluid of animals challenged with cecal contents. These data demonstrate the usefulness of two immunomodulatory polysaccharides in preventing experimental intraabdominal sepsis in the absence of antimicrobial therapy and may represent a new adjunct to antibiotic regimens currently used to prevent clinical cases of this disease.

In humans, endotoxin activates the hypothalamic-pituitary-adrenal (HPA) axis, and the resulting increase in cortisol modulates the immune response. There is little information on the HPA axis response to other antigens. We examined the effect of the protein antigen tetanus toxoid on HPA axis activity in 10 healthy, premenopausal women (aged 28.6 +/- 2.6 yr). Subjects received im injections of placebo and tetanus toxoid at 1600 h on consecutive days. Blood samples for ACTH and cortisol were obtained every half-hour from--1 to 6 h and at 8, 12, and 16 h after each injection. Compared to placebo, tetanus toxoid administration stimulated significant increases in plasma ACTH and serum cortisol, with the maximum cortisol increase of 1.6-fold occurring 4.5 h after drug administration. Urinary free cortisol increased 1.8-fold in the 8 h after tetanus toxoid administration compared to that after placebo administration. Additionally, there was a significant inverse correlation (r = 0.87; P < 0.005) between the tetanus toxoid-induced increase in serum cortisol and the increase in tetanus antibody levels measured 1 month postvaccination. Thus, administration of the protein antigen tetanus toxoid activated the HPA axis in healthy, premenopausal women. This activation of the HPA axis correlated inversely with the antibody response to tetanus toxoid.

Experimental immunoglobulin preparations for treatment of group B streptococcal (GBS) infections contain low levels of functional antibody and exhibit lot-to-lot variability. GBS capsular polysaccharide-protein conjugate vaccines have recently been shown to produce high serum levels of type-specific antibody in healthy volunteers. Treatment of neonatal mice 4 h after inoculation with an ordinarily lethal dose of GBS type Ia, Ib, or III with pooled human serum from adults who had received GBS type Ia capsular polysaccharide-tetanus toxoid vaccine (Ia CPS-TT), Ib CPS-TT, or III CPS-TT resulted in 63%, 70%, and 75% survival, respectively. In contrast, < or = 17% of the infected mice treated with normal human serum or saline survived. These results demonstrate the therapeutic activity of GBS polysaccharide conjugate vaccine-induced antiserum and provide a rationale for the use of these vaccines in producing a functional, high-titered intravenous immunoglobulin preparation for clinical use.

The susceptibility of neonates to invasive infection with type III group B streptococci (GBS) in a radioactive antigen-binding assay (RABA) has been correlated with low maternal serum levels of capsular polysaccharide-specific antibodies. An ELISA was developed using capsular polysaccharide covalently coupled to human serum albumin. In sera from 35 healthy women, the range of IgG antibodies to GBS III polysaccharide was 0.05-33.0 micrograms/mL, and specific IgA antibodies were 0.08-1.1 micrograms/mL; however, no GBS III capsular polysaccharide-specific antibodies of the IgM isotype were detected by the ELISA. The levels of naturally acquired and vaccine-induced antibodies obtained with this ELISA correlated well with the results of the RABA (Spearman's rank correlation coefficient, .92). The ELISA has two major advantages over the RABA: It measures specific isotypes and subclasses of antibodies, and it can detect type III polysaccharide-specific antibodies at lower concentrations.

The capsular polysaccharide complex (CPC) of Bacteroides fragilis exhibits unusual biological properties. This polysaccharide complex promotes the formation of intraabdominal abscesses and, when administered systemically, can prevent abscess induction in a rat model of intraabdominal sepsis. Each of these biological properties is mediated by a T cell-dependent immune mechanism. The CPC consists of two distinct polysaccharides, PS A and PS B, each with repeating units that have positively charged amino groups and negatively charge carboxyl or phosphate groups. Analysis of these polysaccharides as well as of other charged polysaccharides of bacterial origin, before and after chemical modification, revealed that the oppositely charged groups are required for promotion of intraabdominal abscesses as well as for the distinct properties associated with the B. fragilis CPC and delineate one mechanism by which these biological responses occur.

The recent recognition of type V strains as a frequent cause of group B streptococcal (GBS) infection in both infants and adults prompted investigation of an effective vaccine against these organisms. Purified GBS type V polysaccharide was covalently linked to tetanus toxoid to form a type V polysaccharide-tetanus toxoid conjugate vaccine. The conjugate elicited type V polysaccharide-specific IgG antibodies in rabbits, while unconjugated type V polysaccharide did not. Conjugate-induced rabbit antibodies were opsonic in vitro and protected mice against challenge with type V GBS. Efficacy of the conjugate vaccine also was demonstrated in a maternal vaccination/neonatal challenge model in mice. A GBS type V polysaccharide-tetanus toxoid conjugate is an effective immunogen in animal models and may be a useful component for inclusion in a multivalent GBS vaccine for human use.

Neonatal mice were infected with type III group B streptococcal (GBS) strain M781 by the intraperitoneal route. Age-related susceptibility to challenge was seen within the first 5 days of life. Quantitative blood cultures demonstrated a rapid increase in bacterial numbers during the first 30 h after challenge. Infected pups showed clinical signs of septicemia, and most succumbed within 48 h of challenge. Histopathologic evaluation of the neonates showed bacterial infection within 1 day after challenge. Pregnant adult mice were given a single inoculation of serum raised in rabbits against a tetanus toxoid-conjugated type III GBS polysaccharide vaccine. This serum passively protected 100% of the offspring. This neonatal mouse model of GBS infection and protection may be suitable for study of various forms of intervention.

Morphologic study of Bacteroides melaninogenicus subspecies asaccharolyticus by electron microscopy disclosed the presence of a capsule and a cell wall structure otherwise typical of a gram-negative organism. An outer membrane complex was isolated with use of gentle methods. Relative purity of the preparation was confirmed by electron microscopy and by the formation of a single band in a sucrose density gradient. Gel chromatography was used for separation of the major components of the membrane. Antigenicity of the first component, a protein-polysaccharide complex, which cross-reacted with antiserum to another strain of the same subspecies. This component probably represents the capsular antigen and may prove to be the basis for serogrouping. The second membrane fraction differed chemically from the first fraction and represents the lipopolysaccharide component of the outer membrane. Notably, this component lacks 2-keto-3-deoxyoctonate, one of the backbone sugars of aerobic, gram-negative lipopolysaccharides.

A prototype fresh clinical isolate of type Ia group B streptococci (strain 515) can be opsonized by serum containing low levels of antibody. This opsonizing process can also occur in hypogammaglobulinemic serum, confirming its antibody independence, but it does require Ca++ ions and the second component of complement (C2). When formalin-fixed organisms are reacted with whole serum, C4 and C3 are cleaved. These data are strong evidence that this organism can directly activate C1 in the absence of antibody and that this activation leads to the formation of a classical C3 convertase (C4b2a), which cleaves C3. The observation that opsonization can occur in hypogammaglobulinemic serum suggests that complement alone, presumably the fixation of C3b, is sufficient for phagocytes to ingest this pathogen.

Outer membrane material was extracted from a serogroup 1 strain of Legionella pneumophila using ethylenediaminetetraacetate. Ferritin-conjugated antiserum reacted only at the surface of the organism, as seen by electron microscopy. Outer membrane material was fractionated into five peaks by molecular sieve chromatography using a gel that had been equilibrated in a buffer containing sodium deoxycholate. One resolved peak (approximately 4x10(4) daltons) was highly active serologically. When rechromatographed in the absence of sodium deoxycholate, material from this peak reaggregated to approximately 10(6) daltons. Ther serologic activity of this antigen was restricted to L. pneumophila serogroup 2, although minor cross-reactions with strains of serogroups 2 and 4 were detected using an enzyme-linked immunosorbent assay. The antigen was less than 10% carbohydrate, 15% protein, 1.1% phosphate, and the remainder lipid of unknown composition. Neither 2-keto-3-deoxyoctonate nor heptose was detected, and the antigen did not induce a Shwartzman reaction. Only one band was seen on sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

The ability of group B streptococcal (GBS) capsular polysaccharides to potentiate virulence was examined. Incubation of soluble type Ia or III polysaccharide in hypogammaglobulinemic human serum inhibited classical complement-dependent opsonophagocytic killing of type Ia strain 515. When functional complement components were measured, C1 activity increased in sera with added polysaccharide whereas C4 activity decreased 80%-90%. Incubation of purified C1 with type Ia polysaccharide inhibited lysis of EAC4 cells in a C1 transfer assay. In a mouse lethality model, tail-vein injection of 50 micrograms of type Ia or III polysaccharide decreased the 50% lethal dose (LD50) from 3.2 X 10(6) to 2.2 X 10(5). Total hemolytic complement levels in mice immediately after polysaccharide injection were increased over levels in control mice, but this difference was not seen 30 min later. The LD50 in mice depleted of C3 (with cobra venom factor) was 6 X 10(5) and simultaneous injection of polysaccharide did not further lower the LD50.

An indirect fluorescent antibody test was developed with the use of hyperimmune rabbit antiserum to a purified capsular polysaccharide of Bacteroides melaninogenicus subspecies asaccharolyticus. All of 23 strains of B. melaninogenicus subspecies asaccharolyticus were fluorescence-positive in this test. All 11 strains of Bacteroides melaninogenicus subspecies intermedius tested and three strains of Bacteroides melaninogenicus subspecies melaninogenicus were fluorescence-negative. Thirty-one strains of other bacterial species were also fluorescence-negative. The indirect fluorescent antibody test demonstrated the presence of a subspecies-specific capsular antigen from B. melaninogenicus subspecies asaccharolyticus. The capsular antigen was further demonstrated by electron microscopy with ruthenium red, a polysaccharide-staining material.

A sensitive serologic test, the enzyme-linked immunosorbent assay (ELISA), was used to detect serum IgG antibodies directed specifically to a capsular antigen of Bacteroides asaccharolyticus (previously known as Bacteroides melaninogenicus subspecies asaccharolyticus). Anticapsular IgG was measured in 30 specimens of rabbit serum after the animals were immunized with whole B. asaccharolyticus, the two subspecies of B. melaninogenicus, and several other bacterial species. Species-specific anticapsular IgG was demonstrated (P less than 0.001). Levels of anticapsular IgG greater than control levels were likewise detected in serum from two humans, including one patient who had periodontitis and from whom B. asaccharolyticus was isolated, and a laboratory worker who had extensive exposure over a two-year period to B. asaccharolyticus. The ELISA was found to be a relatively simple, sensitive tool for measurement of serum IgG. Its application to detection of immunoglobulins of other classes, including secretory IgA, is anticipated, provided adequate standardization methods are used.

The antibody response to the capsular polysaccharide of Bacteroides fragilis subspecies fragilis in women with acute pelvic inflammatory disease was quantitated using a sensitive radioactive antigen-binding assay. Antibody detected by this technique correlated highly with antibody measured by quantitative precipitin analysis (r = 0.99). Paired acute- and convalescent-phase sera obtained from women from whom B. fragilis subspecies fragilis was isolated by culdocentesis showed significantly greater changes in antibody concentration than corresponding sera obtained from women with gonococcal or nongonococcal pelvic inflammatory disease when B. fragilis subspecies fragilis was not isolated from the cul-de-sac. These data indicate that the steady state of immunity to this capsular antigen is perturbed in patients with acute nongonococcal pelvic inflammatory disease from whom this organism can be cultured. This finding may indicate that B. fragilis can play a significant role in acute pelvic inflammatory disease and can be involved early in the infectious process.

Electron microscopic studies of laboratory-passaged and animal-passaged isolates of Bacteroides fragilis showed significantly more capsular material around the latter. This observation correlated with increased survival of animal-passaged bacteria in rabbit intraperitoneal chambers and increased resistance to phagocytosis and opsonophagocytic killing by neutrophils. With an initial inoculum of 2.5 x 10(6) colony-forming units/ml, the number of bacteria surviving after incubation for 2 hr with neutrophils and pooled normal human serum was significantly (P less than 0.01) greater for animal-passaged than for laboratory-passaged bacteria. Neutrophil uptake of 14C-labeled animal-passaged and laboratory-passaged bacteria after incubation for 20 min was 45% and 63%, respectively (P less than 0.05). No significant difference in survival was found between animal-passaged and laboratory-passaged isolates of Bacteroides thetaiotaomicron. These findings suggest that on mechanism whereby the capsule of B. fragilis contributes to virulence is by inhibiting opsonophagocytic killing.

One hundred three clinical isolates of Bacteroides fragilis were identified during a two-year period. Most of these isolates were strains of B. fragilis subspecies fragilis, which constitutes a minor component of the fecal flora in comparison with the other subspecies of B. fragilis. By use of several techniques for demonstration of capsules, it was found that only B. fragilis strains classified as subspecies fragilis were encapsulated. An indirect immunofluorescence assay was developed for identification of clinical isolates possessing capsular material that was immunologically similar to that found in the reference strain of B. fragilis subspecies fragilis. All strains examined that were classified as subspecies fragilis were positive in this assay for the capsular material, whereas strains of the other subspecies were negative. This tests represents a rapid and sensitive means of identifying the most prevalent anaerobic gram-negative bacillus involved in human infections. The capsular polysaccharide of B. fragilis subspecies fragilis is a unique factor associated with the predominant subspecies of B. fragilis isolated from clinical material.

Strains representing each of the five serologically distinct types of group B Streptococcus and variant strain 090R, which is devoid of type-specific polysaccharide, were studied by electron microscopy after incubation with various ferritin-conjugated antibodies. Ferritin-conjugated types Ia, II and III antibodies localized on homologous cells in a pattern that defines the capsular location of the type Ia, II, and III polysaccharide antigen. Type Ib and Ic cells have surface antigens to which ferritin-labeled antibodies to both capsular polysaccharide and a common surface protein attach.

The pathogenic potentials of encapsulated and unencapsulated strains of Bacteroides fragilis were compared by use of a rat model of intraabdominal sepsis. Implantation of encapsulated B. fragilis alone resulted in abscesses in most recipients, whereas unencapsulated strains seldom produced this effect unless they were combined with another organism. Implants of heat-killed, encapsulated B. fragilis also resulted in abscess formation. Subsequent experiments suggested that the abscess-potentiating ability of encapsulated B. fragilis is related to the capsular polysaccharide. Implantation of 200 microgram of the purified capsular material alone or in conjuction with unencapsulated strains caused abscess formation in a majority of animals. Comparable results were not obtained with capsular polysaccharide from Escherichia coli O7:K1(L)"NM or with heat-killed Streptococcus pneumoniae type III. The capsular polysaccharide of B. fragilis appears to potentiate abscess formation and may represent a virulence factor for this species.

Several bacterial isolated from human pharyngeal cultures specifically inhibited the growth of Legionella pneumophila. The inhibitory substance from two strains (Streptococcus species 1-3 and Staphylococcus saprophyticus KC) was isolated from a broth supernatant. The inhibitor was active against all strains of L. pneumophilia tested, including five strains of L. pneumophila serogroup 1 and one strain each of serogroups 2, 3, and 4. The substance did not inhibit growth of 18 fresh clinical and laboratory pathogens (12 general). The substance was dialyzable, was resistant to head and proteolysis, and did not precipitate with ammonium sulfate. Nicotinamide adenine dinucleotide, produced by several upper respiratory tract organisms, did not inhibit L. pneumophila, and L. pneumophila could not be isolated when Streptococcus species 1-3, S. saprophyticus KC, and L. pneumophila were cocultivated. These properties may in part explain the difficulty of isolation and may aid in the identification of L. pneumophila.